High-performance liquid chromatography of rat and mouse islet polypeptides: potential risk of oxidation of methionine residues during sample preparation.
After preparative high-performance liquid chromatography of mouse islet culture medium, concentrated on disposable C18 cartridges (Sep-Pak), an unexpected insulin immunoreactive peak eluting earlier than mouse insulin I and II was detected. Molecular mass determination by mass spectrometry supported its suspected identity as methionine sulphoxide insulin II. We have examined the formation of Met-O derivatives of insulin II, glucagon and pancreatic polypeptide during sample preparation (Sep-Pak and Speed-Vac concentrating). The oxidation of methionine residues was found to depend very much on the buffer, the organic modifier and the procedure. In particular the use of methanol-trifluoroacetic acid resulted in extensive oxidation. The oxidation could be minimized by adding 2 mM dithiothreitol to the buffer and by degassing and/or nitrogen-bubbling of the buffer. Minimal formation of Met-O derivatives is important for the quantitation of methionine-containing polypeptides.
Linde
S
S
Hagedorn Research Laboratory, Gentofte, Denmark.
Nielsen
J H
JH
Hansen
B
B
Welinder
B S
BS
D000818:Animals; D001205:Ascorbic Acid; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol; D005934:Glucagon / Q000032:analysis; D007328:Insulin / Q000032:analysis; D007515:Islets of Langerhans / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry*; D051379:Mice; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010191:Pancreatic Polypeptide / Q000032:analysis; D010455:Peptides / Q000032:analysis*; D011786:Quality Control; D051381:Rats; D013048:Specimen Handling
D007328:Insulin; D010455:Peptides; D010191:Pancreatic Polypeptide; D005934:Glucagon; D008715:Methionine; D001205:Ascorbic Acid; D004229:Dithiothreitol